The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex (original) (raw)

2023, Nature Communications

The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly. During mitosis, a eukaryotic cell divides into two genetically identical daughter cells. To achieve this, the duplicated chromosomes in the parent cell must be equally distributed into the daughter cells. The spindle assembly checkpoint (SAC) serves as a surveillance mechanism to ensure that duplicated chromosomes are stably attached to spindle microtubules through an adapter structure named the kinetochore. Kinetochores lacking end-on microtubule attachment activate the SAC to prevent premature anaphase onset and avoid chromosome missegregation. The effector molecule generated upon SAC activation is the Mitotic Checkpoint Complex (MCC). The MCC consists of two subcomplexes: BUBR1:BUB3 and CDC20:MAD2 1,2. It inhibits the E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) 3-5. APC/C ubiquitinates Cyclin B1, a key mitosis regulator, thereby targeting it for proteasome-mediated degradation 6-8. Inhibition of the APC/C suppresses the degradation of Cyclin B1, which in turn delays anaphase onset. The formation of the CDC20:MAD2 complex has been identified as the rate-limiting step in the assembly of the MCC 9,10. Other checkpoint proteins, including the MAD1:MAD2 complex and the BUB1:BUB3 complex, catalyze this reaction, by recruiting the MCC subunits at